Synthesis and antimicrobial uses of dinuclear silver(I) pyrazolates

ABSTRACT

Novel dinuclear silver(I) pyrazolido complexes and methods of synthesizing them are provided. Advantageously, the novel silver(I) pyrazolido complexes have excellent antimicrobial activity and methods of using said complexes to treat bacterial, fungal, and viral infections are also provided.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 62/722,664, filed Aug. 24, 2018, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Antibiotic resistance is currently an alarming issue and several healthorganizations throughout the World have already declared bacterialresistance towards antibiotics as “catastrophic”. The Infectious DiseaseSociety of America (IDSA) has expressed significant concern onantibacterial resistance, in particular regarding the multi-drugresistant bacteria (MDR), which have been singled out as an imminentthreat to US public health. Among the Gram-positive variety,methicillin-resistant Staphylococcus aureus (MRSA) is annuallyresponsible for more loss of life in US than the infectious HIV/AIDSdisease. Gram-negative strains have also started showing resistancetowards a range of antimicrobials. The notorious Gram-negative hospitalinfections are mostly caused by Klebsiella pneumonia, Pseudomonasaeruginosa, and Acinetobacter baumannii. These circumstances haveprompted the research community in recent years to focus on developingalternative chemotherapeutics to combat antibacterial resistance.

Ionic or bioactive silver (Ag⁺) has been found to be a quite effectiveantimicrobial against a broad range of microorganisms. Ionic Ag has beenwidely used in various commercially-available healthcare products andalso as an antibacterial agent. Silver is “oligodynamic” because of itsability to exert its antimicrobial actions even at very lowconcentrations. In addition, Ag⁺ ions interact with bacteria by severaldifferent mechanisms, making it difficult for the pathogens to developresistance. In bacteria, Ag⁺ ions interact with the nucleophilic aminoacid residues, inhibit the function of oxidative enzymes, promotegeneration of reactive oxygen species (ROS) and interfere with DNAreplication. These observations have led to silver-based antimicrobials,which are particularly effective against burn wound infections. Forexample, silver nitrate and silver sulfadiazine (SSD) are usedextensively as topical antimicrobials for severe burn wounds. Slow andsustainable delivery of Ag⁺ ions under a physiological milieu bydissociation of the topically applied compound is crucial to itseffective antibacterial action, ensuring also that the Ag-basedantimicrobial is not being over used, which might cause otherundesirable side effects. Appropriately designed Ag(I) coordinationcomplexes can meet these requirements.

Pyrazoles themselves have been reported to exhibit a wide range ofbiological activities, including antimicrobial, anti-fungal,anti-inflammatory, anti-convulsant, anticancer, and neuroprotective.Several pyrazole derivatives have already found their place in someclinically approved non-steroidal anti-inflammatory drugs (NSAIDs).

Many strains of bacteria spores (e.g., Clostridium species),Gram-positive bacteria (e.g., mycobacteria) and Gram-negative bacteria(e.g., Pseudomonas aeruginosa (P. aeruginosa)) have intrinsic resistanceto antimicrobial agents. Moreover, many antimicrobial agents are noteffective against biofilms. For example, Serratia marcescens (S.marcescens) and Burkholderia cepacia (B. cepacia) biofilms are found indisinfectant chlorhexidine solution, P. aeruginosa biofilm in iodophorantiseptics and on the interior surface of polyvinyl chloride pipes usedin the production of providone-iodine antiseptics. Furthermore, overuseof these antimicrobial agents has led to drug resistance in microbes.Major concerns include cross-resistance and co-resistance withclinically used antimicrobial agents, which may present a potentialpublic health risk.

Therefore, there exists a need for antimicrobials that are effectiveagainst a wide range of microbes, are not prone to induce microbialresistance, and provide antimicrobial activity in the context ofbiofilms.

BRIEF SUMMARY OF THE INVENTION

Provided herein are novel dinuclear silver (Ag) (I) pyrazolido complexesand methods of making and using them as antimicrobial agents.Advantageously, the novel dinuclear Ag(I) pyrazolido complexes haveexcellent antimicrobial effects when administered in vitro and in vivo.Further provided are methods of synthesizing the novel dinucelar Ag(I)pyrazolido complexes of the subject invention and methods of using themfor the treatment of microbial infections in subjects in need of suchtreatment and for the growth inhibition and killing of microbes onsurfaces or in compositions of matter such as food or cosmetics.

Advantageously, through introduction of specific ancillary ligands andthe use of water soluble pyrazole, the dinuclear Ag(I) pyrazolidocomplexes of the subject invention can have excellent aqueoussolubility, lipophilicity or both and can be used for excellent cellularuptake and in vitro and in vivo antimicrobial functionality.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show two general formulae of dinuclear silver (I) pyrazolidocomplexes of the invention. FIG. 1A shows abis(3,4,5-R-pyrazolido)tetrakis(1,2-R)disilver(I) complex.

FIG. 1B shows a bis(3,4,5-R-pyrazolido)tetrakis(1,2-R; 1-R)disilver(I)complex. FIG. 1C shows a bis(3,4,5-R-pyrazolido)bis(1-R)disilver(I)complex.

FIG. 2A shows a bis(μ-4-R-pyrazolido)tetrakis(PPh3)disilver(I) complex(1) where R³ and R⁵ are hydrogen and R⁴═R═Cl and complex (3) whenR⁴═R═NO₂.

FIG. 2B shows a bis(μ-4-R-pyrazolido)tris(PPh₃)disilver(I) complex (2)where R³ and R⁵ are hydrogen and R⁴═R=Cl and complex (4) when R⁴═R=NO₂.

FIG. 2C shows a bis(μ-4-Cl-pyrazolido)tetrakis(PTA)disilver(I) complex(5).

FIG. 3 shows comparative ³¹P NMR spectra for complexes (1-5).

FIG. 4 shows a perspective view of the molecular structure of complex(1) where the symmetry equivalent atoms are generated by −x+1, −y, −z+1.

FIG. 5 shows a perspective view of the molecular structure of complex(2), where the symmetry equivalent atoms are generated by −x+1, −y+2,−z+1.

FIG. 6 shows a perspective view of the molecular structure of complex(3).

FIG. 7 shows a perspective view of the molecular structure of complex(4).

FIG. 8 shows a perspective view of the molecular structure of complex(5), where the symmetry equivalent atoms are generated by −x+1, −y,−z+1.

FIG. 9 shows a packing pattern of complex (1) along b axis, with thedotted lines representing weak C—H—Cl intermolecular interactions.

FIG. 10 shows a packing pattern of complex (2) along a axis.

FIG. 11 shows a packing pattern of complex (3) along c axis, with thedotted lines representing weak C—H—O intermolecular interactions.

FIG. 12 shows a packing pattern of complex (4) along a axis, with thedotted lines representing weak C—H—O intermolecular interactions.

FIG. 13 shows a packing pattern of complex (5) along c axis, with thedotted lines representing weak C—H—Cl intermolecular interactions.

FIG. 14 shows a ¹H NMR for complex (1).

FIG. 15 shows a ¹H NMR for complex (2).

FIG. 16 shows a ¹H NMR for complex (3).

FIG. 17 shows a ¹H NMR for complex (4).

FIG. 18 shows a ¹H NMR for complex (5).

FIG. 19 shows a IR spectrum for complex (1).

FIG. 20 shows a IR spectrum for complex (2).

FIG. 21 shows a IR spectrum for complex (3).

FIG. 22 shows a IR spectrum for complex (4).

FIG. 23 shows a IR spectrum for complex (5).

FIGS. 24A-24H show Pseudomonas aeruginosa lawns after 18 h incubationwith pellets of 2% (w/w) of complex (5) (FIG. 24A), AgNO3 (FIG. 24B),PTA (FIG. 24C), 4-Cl-pzH (FIG. 24D), complex (1) (FIG. 24E), complex (2)(FIG. 24F), PPh3 (FIG. 24G) and blank plate (FIG. 24H).

FIG. 25 shows abis(3,4,5-R-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complex (6) wherein R³ and R⁵ are hydrogen and R⁴═R can beH. Cl, or NO₂.

FIG. 26 showsbis(3,4,5-R-pyrazolido)tetrakis(phosphaadamantane)disilver(I) complex(7) wherein R³ and R⁵ hydrogen and R⁴═R can be H. Cl, or NO₂.

FIG. 27 shows abis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complex (8).

FIG. 28 shows abis(μ-4-hydroxyethyl-pyrazolido)tetrakis(1,3,5-triazaphospha-adamantane)disilver(I) complex (9).

FIG. 29 shows abis(μ-4-sulfonylchloride-pyrazolido)tetrakis(1,3,5-triazaphospha-adamantane)disilver(I) complex (10).

FIG. 30 shows abis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(phospha-adamantane)disilver(I) complex (11).

FIG. 31 shows a bis(μ-4-hydroxyethyl-pyrazolido)tetrakis(phosphaadamantane)disilver(I) complex (12).

FIG. 32 shows a bis(μ-4-sulfonylchloride-pyrazolido)tetrakis(phosphaadamantane)disilver(I) complex (13).

FIG. 33 shows abis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I) complex (14).

FIG. 34 shows a bis(μ-4-hydroxyethyl-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I) complex (15).

FIG. 35 shows abis(μ-4-sulfonylchloride-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I) complex (16).

FIG. 36 shows abis(μ-4-adamantane-imine-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I) complex (17).

FIG. 37 shows abis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complex (18).

FIG. 38 shows a bis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis(phosphaadamantane)disilver(I) complex (19).

FIG. 39 shows abis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis((4-sulfophenyl)-diphenylphosphine)disilver(I) complex (20).

FIG. 40 shows abis(μ-4-adamantane-amine-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I) complex (21).

FIG. 41 shows abis(3,4-R-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complex (22)wherein R³ and R⁵ are hydrogen and R⁴═R can be H. Cl, or NO₂.

FIG. 42 shows a bis(3,4,5-R-pyrazolido)tetrakis(phenanthrenediphenylphosphine)disilver(I) complex (23) wherein R³ and R⁵ arehydrogen and R⁴═R can be H. Cl, or NO₂.

FIG. 43 shows abis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complex (24).

FIG. 44 shows abis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis((8-(4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complex (25).

FIG. 45 shows abis(μ-4-hydroxyethyl-pyrazolido)tetrakis((8-(4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complex (26).

FIG. 46 shows abis(μ-4-sulfonylchloride-pyrazolido)tetrakis((8-(4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complex (27).

FIG. 47 shows abis(μ-4-chloride-pyrazolido)bis(1,3,5-triazaphosphaadamantane)disilver(I) complex (28).

FIG. 48 shows a perspective view of the molecular structure of complex(28).

FIG. 49 shows abis(μ-3-methyl-pyrazolido)bis(1,3,5-triazaphosphaadamantane) disilver(I)complex (29).

FIG. 50 shows a perspective view of the molecular structure of complex(29).

FIGS. 51A-51B show the results of bacterial eradication by compounds ofcomplex (5) and complex (29) assessed using the optical density method.FIG. 51A shows the optical density of P. aeruginosa treated withincreasing concentrations of complex (5). FIG. 51B shows the opticaldensity of P. aeruginosa treated with increasing concentrations ofcomplex (29).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods of synthesis and use ofnovel Ag(I) pyrazolido complex-based antimicrobial compounds that areeffective against a broad spectrum of bacteria, viruses, and fungi; donot induce microbial resistance; and provide anti-microbial activityeven in the context of complex microbial structures, including biofilms.

The novel silver Ag(I) pyrazolido complexes of the subject inventionenable slow and sustainable delivery of Ag⁺ ions under physiologicalconditions and provide excellent antibacterial activity in vitro and invivo with no inadvertent toxicity due to release of Ag⁺ underphysiological conditions.

In some embodiments, the invention provides novel silver(I) pyrazolidocomplexes of the general formula of structure (A):

wherein R¹ and R² are each independently selected from the groupconsisting of 1,3,5-triaza-7-phosphaadamantane (PTA), phosphaadamantane(PA), P(R⁶)₃, (4-sulfophenyl) diphenyl-phosphine (SDPP), phenanthrenediphenylphosphine (PDPP),8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PMBODIPY),8-((4-phosphino)phenyl)-4,4-diethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PEBODIPY), and8-((4-phosphino)phenyl)-4,4-diphenyl-1,3,5,7-tetramnethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PPBODIPY);m is an integer selected from the group consisting of 0 and 1;n is an integer selected from the group consisting of 0 and 1;wherein R³, R⁴, and R⁵ are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, nitro, alkyl, alkenyl, aryl,formyl, acetyl, hydroxyalkyl, halogen substituted alkyl, halogensubstituted aryl, halogen substituted sulfuryl, imine-linked PTA,amine-linked PTA, imine-linked adamantane, amine-linked adamantine,imine-linked triphenylphosphine, and amine-linked triphenylphosphine;and each R⁶ is independently selected from the group consisting of alkyland aryl.

In some embodiments, the invention provides novel silver(I) pyrazolidocomplexes of the general formula of structure (C):

wherein R¹ and R² are each independently selected from the groupconsisting of 1,3,5-triaza-7-phosphaadamantane (PTA), phosphaadamantane(PA), (PR⁶)₃, (4-sulfophenyl) diphenyl-phosphine (SDPP), phenanthrenediphenylphosphine (PDPP),8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PMBODIPY),8-((4-phosphino)phenyl)-4,4-diethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PEBODIPY), and8-((4-phosphino)phenyl)-4,4-diphenyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PPBODIPY);

m is an integer selected from the group consisting of 0 and 1;

n is an integer selected from the group consisting of 0 and 1;

wherein R³, R⁴, and R⁵ are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, nitro, alkyl, alkenyl, aryl,formyl, acetyl, hydroxyalkyl, halogen substituted alkyl, halogensubstituted aryl, halogen substituted sulfuryl, imine-linked PTA,amine-linked PTA, imine-linked adamantane, amine-linked adamantine,imine-linked triphenylphosphine, and amine-linked triphenylphosphine;wherein each R⁶ is independently selected from the group consisting ofalkyl and aryl; or a pharmaceutically acceptable salt thereof.

In some embodiments, methods are provided that comprise reacting4-Cl-pyrazole with silver benzoate (Ag(PhCOO)) in equimolar amounts ofdry Tetrahydrofuran (THF) to generate insoluble[Ag(4-Cl-pyrazolate)]_(n) polymers, which polymers are subsequentlyreacted with excess of triphenylphosphine (PPh₃) to generate the dimericsilver complex disilver(I)-bis(μ-4-Cl-pyrazolido) tetrakis(PPh₃)(complex (1)).

In other embodiments, methods are provided that comprise reacting4-Cl-pyrazole with silver benzoate (Ag(PhCOO)) in equimolar amounts ofdry THF to generate insoluble [Ag(4-Cl-pyrazolate)]_(n) polymers, whichpolymers are subsequently reacted with 1.5 equivalent oftriphenylphosphine (PPh₃) to generate the dimeric silver complexdisilver(I)-bis(μ-4-Cl-pyrazolido) (PPh₃)₃ (complex (2)).

In further embodiments, methods are provided that comprise reacting4-NO₂-pyrazole with silver benzoate to generate[Ag(4-NO₂-pyrazolate)]_(n) polymers that are subsequently reacted withexcess of triphenylphosphine to generate the dimeric silver complexdisilver(I)-bis (μ-4-NO₂-pyrazolido)tetrakis(PPh₃) (complex (3)).

In yet other embodiments, methods are provided that comprise reacting4-NO₂-pyrazole with silver benzoate to generate[Ag(4-NO₂-pyrazolate)]_(n) polymers that are subsequently reacted with1.5 equivalents of triphenylphosphine to generate the dimeric silvercomplex disilver(I)-bis (μ-4-NO₂-pyrazolido)(PPh₃)₃].4H₂O (complex (4)).

In some embodiments, the methods of the subject invention comprisereacting 4-Cl-pyrazole with silver benzoate to generate insoluble[Ag(4-Cl-pyrazolate)]_(n) polymers that are subsequently reacted with 4equivalents of 1,3,5-triazaphosphaadamantane (PTA) to generate thedimeric silver complex disilver(I)-bis(μ-4-Cl-pyrazolido)tetrakis(PTA)(complex (5)).

In preferred embodiments, the methods of the subject invention comprisereacting 4-Cl-pyrazole with silver benzoate to generate insoluble[Ag(4-Cl-pyrazolate)]_(n) polymers that are subsequently reacted with 2equivalents of 1,3,5-triazaphosphaadamantane (PTA) to generate thedimeric silver complex disilver(I)-bis(μ-4-chloride-pyrazolido)bis(PTA)(complex (28)).

In further preferred embodiments, the methods of the subject inventioncomprise reacting 3-Methyl-pyrazole with silver benzoate to generateinsoluble [Ag(3-Me-pyrazolate)]_(n) polymers that are subsequentlyreacted with 2 equivalents of 1,3,5-triazaphosphaadamantane (PTA) togenerate the dimeric silver complexdisilver(I)-bis(μ-3-methyl-pyrazolido)bis(PTA) (complex (29)).

The symmetrical or unsymmetrical dinuclear Ag(I) pyrazolido complexesgenerated, according to the methods of the subject invention, haveadvantageous in vitro and in vivo functions including, but not limitedto, antibacterial, antifungal, antiviral, anti-inflammatory,anti-convulsant, anti-cancer, neuroprotective, angiotensin convertingenzyme (ACE) inhibitory, cholecystokinin-1 receptor antagonistic, and/orfor estrogen receptor ligand activity.

The symmetrical dinuclear Ag(I) pyrazolido complexes can comprise twophosphine groups on each silver atom or, alternatively, can comprise onephosphine group on each silver atom.

The Ag(I) pyrazolido complexes, according to the subject invention, canbe modified by introducing functional groups including, but not limitedto, halogen, nitro, alkyl, alkenyl, aryl, formyl, acetyl, halogensubstituted alkyl, and/or halogen substituted aryl in pyrazole positions3-, 4-, and 5- to modulate aqueous solubility.

According to specific embodiments of the subject invention, thesymmetrical and unsymmetrical Ag(I) pyrazolido complexes of the subjectinvention are further modified to enhance their aqueous solubility,lipophilicity, or both, by introducing appropriate ancillary groups.

In some embodiments, the dinuclear Ag(I) pyrazolido complexes are highlylipophilic.

In preferred embodiments, the dinuclear Ag(I) pyrazolido complexes ofthe subject invention are highly soluble in water. In more preferredembodiments, the dinuclear Ag(I) pyrazolido complexes of the subjectinvention possess optimal combinations of hydrophilic and lipophilicligands to enable excellent cellular uptake and low toxicity.

An increased aqueous solubility can be imparted on the dinuclear Ag(I)pyrazolido complexes of the subject invention, using several strategies.

First, introduction of 1,3,5-triazaphosphaadamantane (PTA) ligands intothe dinuclear Ag(I) pyrazolido complex, e.g., in complexes (5) and (6),results in substantially increased aqueous solubility due to strongH-bond interaction of the bridgehead N atoms with water molecules.

Second, increased aqueous solubility is accomplished by employing bothwater-soluble pyrazole derivatives and ancillary ligands. Examples ofdinuclear Ag(I) pyrazolido complexes generated according to thisstrategy include, but are not limited to, complexes (8), (9), (10),(14), (15), and (16).

Third, increased aqueous solubility is accomplished by utilizingwater-soluble pyrazole and highly lipophilic ancillary ligands. Examplesof dinuclear Ag(I) pyrazolido complexes generated according to thisstrategy include, but are not limited to, complexes (11), (12), (13),and (17). Advantageously, it was found that the latter strategyproviding Ag(I) pyrazolido complexes having an optimized combination ofhydrophilic and lipophilic groups provide unexpected superiorperformance in vitro and in vivo. Without wanting to be bound by theory,it is indicated that the high physiological acceptability of suchcomplexes combined with the optimal lipophilicity for improved cellularuptake causes their unexpected superior in vitro and in vivoperformance.

In further embodiments, dinuclear Ag(I) pyrazolido complexes comprisingan amine form of the pyrazole derivative are provided, which complexesalso have high water solubility. Examples of such complexes include, butare not limited to, complexes (18), (19), (20), and (21).

Fluorescent reporter molecules, in general, allow the detection of thelocation of compounds comprising said reporters following in vitroand/or in vivo administration.

Also provided herein are fluorescent reporter-containing Ag(I)pyrazolido complexes and methods of synthesizing highly lipophilicbiocompatible fluorescent reporter-containing dinuclear Ag(I) pyrazolidocomplexes including, but not limited to, complexes (22) and (23).

In preferred embodiments, methods are provided for synthesizing watersoluble dinuclear Ag(I) pyrazolido complexes incorporating biocompatiblefluorescent reporters, including, but not limited to, complexes (24),(25), (26), and (27). Advantageously, the complexes (24) to (27) provideunexpected superior performance in vitro and in vivo. Without wanting tobe bound by theory, it is indicated that the superior functionality ofcomplexes (24) to (27) is based on the optimized combination ofhydrophilic and lipophilic groups providing high physiologicalacceptability of the complexes combined with the optimal lipophilicityfor improved cellular uptake.

In preferred embodiments, methods are provided for making the dinuclearAg(I) pyrazolido complexes of the subject invention by introducingancillary ligands. In preferred embodiments, dinculear Ag(I) pyrazlidocomplexes are synthesized using [Ag₂O] (silver oxide), or [AgL]precursors, where L=CH₃COO, PhCOO, CF₃SO₃, NO₃, Cl (silver acetate,silver benzoate, silver trifluorosulfonate, silver nitrate, silverchloride, respectively). The pyrazole derivatives employed in themethods of the invention have different groups at the 3-, 4-,5-positions, R³, R⁴, and R⁵, which are independently selected from thegroup consisting of hydrogen, halogen, hydroxyl, nitro, alkyl, alkenyl,aryl, formyl, acetyl, hydroxyalkyl, halogen substituted alkyl, halogensubstituted aryl, halogen substituted sulfuryl, imine-linked PTA,amine-linked PTA, imine-linked adamantane, amine-linked adamantine,imine-linked triphenylphosphine, and amine-linked triphenylphosphine.

Furthermore, the R¹ and R² groups of the pyrazole derivatives areindependently selected from the group consisting of1,3,5-triaza-7-phosphaadamantane (PTA), adamantane, phosphaadamantane(PA), P(R⁶)₃, where the R⁶ groups are selected from alkyl and aryl,(4-sulfophenyl)diphenylphosphine (SDPP), phenanthrene diphenylphosphine(PDPP),8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PMBODIPY),8-((4-phosphino)phenyl)-4,4-diethyl-1,3,5,7-tetramethyl2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PEBODIPY), and8-((4-phosphino)phenyl)-4,4-diphenyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene:PPBODIPY.

The pyrazole derivatives are reacted with the silver precursors inequimolar ratio in dichloromethane or chloroform at ambient conditionsto generate polymeric species with general formula, [Ag(pz*)]n (wherepz*=differently substituted pyrazolido anions). Subsequently thesepolymeric compounds are reacted with phosphines, including but notlimited to P(R⁶)₃, where the R⁶ groups are selected from alkyl and aryl,PTA, PA, sulfophenyl)diphenylphosphine (SDPP), phenanthrenediphenylphosphine (PDPP), and/or a primary fluorescent phosphine BODIPY,including, but not limited to, PMBODIPY, PEBODIPY, and PPBODIPY togenerate the desired dinuclear Ag(I) pyrazolido complexes of theinvention.

For example, complex (5) of the subject invention comprising1,3,5-triazaphosphaadamantane (PTA) as a co-ligand is highlywater-soluble and provides unexpected superior antibacterial properties.Without wanting to be bound by theory it is submitted that by virtue ofthe ability of the PTA ligands of complex (5) of the invention to H-bondstrongly with water molecules through the bridgehead N atoms, thedinucelar Ag(I) pyrazolido complex (5) of the invention is highlybiocompatible and has minimal intrinsic toxicity.

Also provided are methods of synthesizingbis(μ-4-R-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane) disilver(I)complexes, e.g., of complex (6) wherein R can be H, Cl, or NO₂.

In some embodiments, methods are provided to synthesizebis(μ-4-R-pyrazolido) tetrakis(phosphaadamantane)disilver(I) complexes,e.g., of complex (7) wherein R can be H, Cl, or NO₂.

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complexes, e.g., ofcomplex (8).

In some embodiments, methods are provided to synthesizebis(μ-4-hydroxyethyl-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complexes, e.g., ofcomplex (9).

In some embodiments, methods are provided to synthesizebis(μ-4-sulfonylchloride-pyrazolido)tetrakis(1,3,5-triazaphosphaadamantane)disilver(I) complexes, e.g.,complex (10).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(phosphaadamantane)disilver(I)complexes, e.g., complex (11).

In some embodiments, methods are provided to synthesizebis(μ-4-hydroxyethyl-pyrazolido)tetrakis(phosphaadamantane)disilver(I)complexes, e.g., complex (12).

In some embodiments, methods are provided to synthesizebis(μ-4-sulfonylehloride-pyrazolido)tetrakis(phosphaadamantane)disilver(I)complexes, e.g., complex (13).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis((4-sulfophenyl)-diphenylphosphine)disilver(I)complexes, e.g., complex (14).

In some embodiments, methods are provided to synthesizebis(μ-4-hydroxyethyl-pyrazolido)tetrakis((4-sulfophenyl)-diphenylphosphine)disilver(I)complexes, e.g., complex (15).

In some embodiments, methods are provided to synthesizebis(μ-4-sulfonylehloride-pyrazolido)tetrakis((4-sulfophenyl)-diphenylphosphine)disilver(I)complexes, e.g., complex (16).

In some embodiments, methods are provided to synthesizebis(μ-4-adamantane-imine-pyrazolido)tetrakis((4-sulfophenyl)-diphenylphosphine)disilver(I)complexes, e.g., complex (17).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5,-triazaadamantane-amine-pyrazolido)tetrakis(PTA)disilver(I)complexes, e.g., complex (18).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis(phosphaadamantane)disilver(I)complexes, e.g., complex (19).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I)complexes, e.g., complex (20).

In some embodiments, methods are provided to synthesizebis(μ-4-adamantane-amine-pyrazolido)tetrakis((4-sulfophenyl)diphenylphosphine)disilver(I)complexes, e.g., complex (21).

In some embodiments, methods are provided to synthesizebis(μ-4-R-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complexes, e.g., complex (22) wherein R can be H, Cl, orNO₂.

In some embodiments, methods are provided to synthesizebis(μ-4-R-pyrazolido)tetrakis(phenanthrene-diphenylphosphine)disilver(I) complex (23) whereinR can be H, Cl, or NO₂.

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-imine-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,b 4a-diaza-s-indacene)disilver(I) complexes, e.g., complex (24).

In some embodiments, methods are provided to synthesizebis(μ-4-1,3,5-triazaadamantane-amine-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complexes, e.g., complex (25).

In some embodiments, methods are provided to synthesizebis(μ4-hydroxyethyl-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver(I) complexes, e.g., complex (26).

In some embodiments, methods are provided to synthesizebis(μ-4-sulfonylchloride-pyrazolido)tetrakis(8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene)disilver (I) complexes, e.g., complex (27).

In some embodiments, methods are provided to synthesizebis(μ-4-chloride-pyrazolido)bis(1,3,5-triazaphosphaadamantane)disilver(I) complexes, e.g., complex(28).

In some embodiments, methods are provided to synthesizebis(μ-3-methyl-pyrazolido) bis(1,3,5-triazaphosphaadamantane)disilver(I)complexes, e.g., complex (29).

Also provided are methods of using compositions, such methods cancomprise treating bacterial infections in subjects in need of suchtreatment. For example, the methods of treating a subject at risk of, orhaving, a bacterial, fungal infection, and/or viral infection compriseadministering to the subject a composition according to the subjectinvention, which composition comprises a dinuclear silver(I) pyrazolidocomplex of the invention.

Further provided are methods of killing microbes and/or inhibiting theirgrowth, the method comprising contacting the microbes with a compositionaccording to the subject invention, which composition comprises adinuclear silver(I) pyrazolido complex of the invention.

For example, in a preferred embodiment, using a composition comprisingcomplex (5), an unexpected antibacterial effect against theGram-negative bacterial strain P. aeruginosa was observed.

In further embodiments, it was surprisingly determined that acomposition comprising complex (29) resulted in a significantantibacterial effect against the Gram-negative bacterial strain P.aeruginosa, which antibacterial effect was significantly higher than theantibacterial effect of a composition comprising, e.g., complex (5).

In some embodiments of the subject invention, the Ag(I) pyrazolidocomplexes provided are highly active against Gram-positive microbes(e.g., S. aureus), Gram-negative microbes (e.g., Escherichia coli (E.coli), P. aeruginosa), fungi (e.g., C. albicans), and viruses and can beused in methods of treating subjects suffering from such Gram-positive,Gam-negative, fungal, and viral infections.

Advantageously, the Ag(I) pyrazolido complexes of the subject inventionare biocompatible, non-hemolytic, and non-cytotoxic at concentrationsabove the minimum inhibitory concentration (MIC), and are thereforeattractive for a wide range of consumer products such as, for example,cosmetics, skin lotions or creams, antibiotic drugs, food preservatives,surface cleaners, antiseptic agents, and/or wound care products. Thecompositions can also be used to control plant and animal pathogens. A“biocompatible” material is defined herein as a material capable ofperforming with an appropriate host response in a specific application.

Minimum inhibitory concentration (MIC) is defined as the minimumconcentration of a composition required to inhibit growth of a givenmicrobe for an 18 hour period (bacteria) or 42 hour period (fungi,viruses). A MIC less than 1 mM is desirable. Even more desirable is aMIC of 10 μM or less. A lower MIC indicates higher antimicrobialactivity.

Minimum bactericidal concentration (MBC) is defined as the minimumconcentration of a composition required in order to kill a givenmicrobe. A lower MBC indicates higher antimicrobial activity.

The complexes of the subject invention can have a minimum inhibitoryconcentration (MIC) of about 100 mM to about 0.1 nM and preferably about10 mM to about 0.1 nM, and more preferably 1 μM to about 0.1 nM againsta bacterium. In a specific embodiment, the complexes can have a MIC ofabout 5 μM to about 5 nM against P. aeruginosa.

Non-limiting exemplary bacteria that can be inhibited or killed withAg(I) pyrazolido complexes, according to the subject invention, include,but are not limited to Gram-positive Staphylococcus aureus (S. aureus),Gram-negative Escherichia coli (E. coli), fungus Candida albicans (C.albicans) and other yeasts and Gram-negative Pseudomonas aeruginosa (P.aeruginosa). Other microbes include Gram-positive Staphylococcusepidermidis (S. epidermidis), Gram-positive Methicillin-resistantStaphylococcus aureus (MRSA), Gram-positive Vancomycin-resistantEnterococcus (VRE), Gram-negative Acinetobacter baumannii (A.baumannii), Gram-negative Klebsiella pneumoniae (K. pneumoniae) and thefungus Cryptococcus neoformans (C. neoformans).

Further Gram-positive bacteria to be treated according to the subjectinvention include, but are not limited to, Actinomyces, Arthrobacter,Bifidobacterium, Corynebacteruim, Frankia, Micrococcus, Micromonospora,Mycobacterium, Nocardia, Propionibacterium, Streptomyces, Bacillus,Listeria, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Leuconostoc, Pediococcus, Streptococcus, Acetobacterium, Clostridium,Eubacterium, Heliobacterium, Megasphaera, Pectinatus, Selenormonas,Zymophilus, Sporomusa, Mycoplasma, Spiroplasma, Ureaplasma, andErysipelothrix.

Further Gram-negative bacteria to be treated according to the subjectinvention include, but are not limited to, Acinetobacter,Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria,Enterobacter, Erwinia, Escherichia coli, Franciscella, Helicobacter,Hemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella,Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio,and Yersinia.

Further fungi to be treated according to the subject invention include,but are not limited to, Coccidioides, Cryptococcus gatii, Pneumocystisjirovecii, Sporothrix, Blastomyces, Cryptococcus neofromans,Histoplasma, Talaromyces, and Tinea corporis.

Furthermore, viruses to be treated according to the subject invention,include, but are not limited to, Herpes simplex virus type 1 and 2,Human Immunodeficiency Virus (HIV), Hepatitis B virus, Hepatitis Cvirus, Rhinovirus, Influenza virus, Variola virus, Human enterovirus C,Cowpox virus, Respiratory syncytial virus, Paramyxoviridiae, Poxviridae,and Picornaviridae.

The Ag(I) pyrazolido complexes, according to the subject invention, canbe provided separately or in combination as medicaments that areantibacterial, antiviral, antifungal, or any combination thereof. Themedicaments can be formulated according to known methods for preparingpharmaceutically useful compositions. Such pharmaceutical compositionscan be adapted for various forms of administration, such as, but notlimited to, oral, parenteral, intravenous, nasal, topical, pulmonary,and transdermal. The Ag(I) pyrazolido complexes, according to thesubject invention, can be provided as solutions, amorphous compounds,injectables, pills, inhalants, or in any other form for administration.The compositions comprising Ag(I) pyrazolido complexes of the subjectinvention can include pharmaceutically acceptable carriers or diluents.Formulations are described in a number of sources, which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin E W [1995] Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations that can be usedin connection with embodiments of the invention.

Formulations suitable for administration include, e.g., aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes, which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, or tablets of the Ag(I) pyrazolido complexcomprising compositions. It should be understood that in addition to theingredients particularly mentioned above, the formulations of thesubject invention can include other agents conventional in the arthaving regard to the type of formulation in question.

Pharmaceutically acceptable carriers used in Ag(I) pyrazolido complexformulations according to the subject invention include, but are notlimited to, inert diluents and vehicles such as: one or more excipients,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and aerosol sprays. Tablets, troches, pills, capsules, and the like may,but do not necessarily, contain binders, such as gum tragacanth, acacia,corn starch or gelatin; excipients, such as dicalcium phosphate;disintegrating agents, such as corn starch, potato starch, or alginicacid; lubricants, such as magnesium stearate; sweetening agents, such assucrose, fructose, lactose or aspartame; flavoring agents, such aspeppermint, oil of wintergreen, or cherry flavoring; liquid carriers,such as a vegetable oil or a polyethylene glycol; and/or solid carriers;such as finely divided solids such as talc, clay, microcrystallinecellulose, silica, or alumina. Any material used in preparing the dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. The dosage form may be a sustained-releasepreparation. Other dosage forms can include surfactants or otheradjuvants. Liquid compositions for topical use can be applied fromabsorbent pads or be impregnated on bandages and other dressings.Thickeners, such as synthetic polymers, fatty acids, fatty acid saltsand esters, fatty alcohols, modified celluloses or modified mineralmaterials, can be employed with liquid carriers.

Ag(I) pyrazolido complexes may be in the free base form or in the formof an acid salt thereof. In some embodiments, compounds as describedherein may be in the form of a pharmaceutically acceptable salt, whichare known in the art (Berge S. M. et al., J. Pharm. Sci. (1977)66(1):1-19). Pharmaceutically acceptable salts as used herein include,for example, salts that have the desired pharmacological activity of theparent compound (salts which retain the biological effectiveness and/orproperties of the parent compound and which are not biologically and/orotherwise undesirable). The acid salts can be generated with anypharmaceutically acceptable organic or inorganic acid.

Pharmaceutically acceptable salts may be derived from, e.g., and withoutlimitation, acetic acid, adipic acid, alginic acid, aspartic acid,ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid,cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid,cyclopentanepropionic acid, diethylacetic acid, digluconic acid,dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid,glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolicacid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloricacid, hydrobromic acid, hydriodic acid, 2-hydroxyethanesulfonic acid,isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid,mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid,naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid,nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionicacid, phosphoric acid, picric acid, pimelic acid, pivalic acid,propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuricacid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid.Salts, as described herein, may be prepared by conventional processesknown to a person skilled in the art, for example, and withoutlimitation, by combining the free form with an organic acid or cationexchange from other salts. Those skilled in the art will appreciate thatpreparation of salts may occur in situ during isolation and purificationof the compounds or preparation of salts may occur by separatelyreacting an isolated and purified compound.

Silver(I) pyrazolido complexes or pharmaceutical compositions for use asdescribed herein may be administered by means of a medical device orappliance such as an implant, graft, prosthesis, stent, etc. Also,implants may be devised that are intended to contain and release suchcompounds or compositions. An example would be an implant made of apolymeric material adapted as a vehicle to release the Ag(I) pyrazolidocomplexes over a period of time.

An “effective amount” of a Ag(I) pyrazolido complex comprisingpharmaceutical composition includes a therapeutically effective amountor a prophylactically effective amount. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of a Ag(I) pyrazolido complexformulation may vary according to factors such as the disease state,age, sex, and weight of the subject, and the ability of the compound toelicit a desired response in the subject. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the compound are outweighed by the therapeutically beneficialeffects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, a prophylactic dose is used in subjectsprior to the disease, so that a prophylactically effective amount may beless than a therapeutically effective amount.

Dosage values may vary with the severity of the condition to bealleviated. For any particular subject, specific dosage regimens may beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions. Dosage ranges suggested herein are exemplary onlyand do not limit the dosage ranges that may be selected by medicalpractitioners. The amount of Ag(I) pyrazolido complexes in thecomposition may vary according to factors such as the disease state,age, sex, and weight of the subject. Dosage regimens may be adjusted toprovide the optimum therapeutic response.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Materials and Methods

Reagent grade chemicals were purchased from Aldrich Chemical Co, AlfaAesar, Fisher scientific and Acros Organics. THF was distilled overNa/benzophenone and CH₂Cl₂ was distilled over CaCl₂/CaH₂ prior to use.NMR spectra were recorded on 400 MHz or 500 MHz Bruker Avancespectrometers. FT-IR spectra were recorded with a Perkin Elmer Spectrum100 FT-IR Spectrometer. Elemental analyses (CHN) were performed byGalbraith Laboratories, Inc. at Knoxville, Tenn. The detail proceduresfor preparations of all complexes are included in the SupplementaryInformation. The IR and ¹H NMR spectra of some of the complexes havealso been provided.

Example 1—Synthesis and Structures of the Complexes

Reaction of 4-R-pzH (where R═Cl and NO₂ and pzH=pyrazole) with silverbenzoate in equimolar amounts in dry THF at room temperature uniformlyresulted in insoluble polymers, [Ag(R-pz)]n. These polymeric complexeswere isolated and suspended in CH₂Cl₂ solution and reaction withappropriate phosphines (PPh₃ and PTA) at ambient temperature resulted inthe corresponding dimeric silver complexes (1) to (5). Depending on theamount of the phosphine used in these reactions, either symmetrical orunsymmetrical dinuclear complexes were isolated. For example, when[Ag(R-pz)]n polymers were reacted with 1.5 equivalents per Ag atom of,e.g., triphenylphosphine (PPh₃) or 1.5 equivalents per Ag atom of PTA,dimeric silver complexes were generated with two PPh₃ groups on a firstsilver atom and one PPH₃ group on a second silver atom or, with two PTAgroups on a first silver atom and one PTA group on a second silver atom.

When [Ag(R-pz)]n polymers were reacted with 1 equivalent per Ag atom of,e.g., triphenylphosphine (PPh₃) or 1 equivalent per Ag atom of PTA,dimeric silver complexes were generated with one PPh₃ group on eachfirst and second silver atom or one PTA on each first and second silveratom.

When [Ag(R-pz)]n polymers were reacted with 2 equivalents per Ag atomof, e.g., triphenylphosphine (PPh₃) or 2 equivalents per Ag atom of PTA,dimeric silver complexes were generated with two PPh₃ groups on eachfirst and second silver atom or two PTA groups on each first and secondsilver atom.

All complexes provided herein were neutral and soluble in variety oforganic solvents. Moreover, complex (5) was also highly soluble inaqueous media, see: S. Kandel, J. Stenger-Smith, I. Chakraborty, R. G.Raptis, Syntheses and X-ray crystal structures of a family of dinuclearsilver(I)pyrazolates: Assessment of their antibacterial efficacy againstP. aeruginosa with a soft tissue and skin infection model, Polyhedron(2018) 154, 390-397, (doi: https://doi.org/10.1016/j.poly.2018.08.015)incorporated herewith in its entirety.

Further complexes, according to the subject invention, synthesized using[Ag₂O] (silver oxide), or [AgL] precursors, where L=CH₃COO, PhCOO,CF₃SO₃, NO₃, Cl (silver acetate, silver benzoate, silvertrifluorosulfonate, silver nitrate, silver chloride, respectively).

Three pyrazole derivatives with different groups at 3-, 4-, 5-positions(R³, R⁴, and R⁵, respectively, see Structure B below, were employed.Reactions of these pyrazoles with the silver precursors in 1, 1.5, or 2equivalents in dichloromethane or chloroform at ambient conditionsfurnish polymeric species with general formula, [Ag(pz*)]_(n) (wherepz*=differently substituted pyrazoles). The subsequent reactions ofthese polymeric compounds with appropriate tertiary phosphines P(R⁶)₃,where the R⁶ groups are selected from alkyl and aryl, PTA, PA, SDPP,PDPP, and/or a primary fluorescent phosphine BODIPY PMBODIPY, PEBODIPY,and PPBODIPY generated the desired complexes.

wherein R³, R⁴, and R⁵ are independently selected from the groupconsisting of halogen, hydroxyl, nitro, alkyl, alkenyl, aryl, formyl,acetyl, hydroxyalkyl, halogen substituted alkyl, halogen substitutedaryl, halogen substituted sulfuryl, imine-linked PTA, amine-linked PTA,imine-linked adamantane, amine-linked adamantine, imine-linkedtriphenylphosphine, and amine-linked triphenylphosphine; and each R⁶ isindependently selected from the group consisting of alkyl and aryl.

Example 2—Synthesis of [Ag₂(4-Cl-pz)₂(PPh₃)₄] Complex (1)

Amounts of 10.3 mg (0.101 mmol) 4-Cl-pzH and 22.9 mg (0.10 mmol)Ag(PhCOO) were added to 5 mL THF and the reaction mixture was stirred atroom temperature for 12 h. After this time, the mixture was filtered andthe insoluble polymeric [Ag(4-Cl-pz)]_(n) was collected by filtrationand was thoroughly washed with CH₂Cl₂ to remove any unreacted startingmaterials. The solid was then dried under vacuum (Yield, 19.5 mg (93%),based on Ag(PhCOO) as a limiting reagent). Subsequently, 19.5 mg (0.093mmol) of the dry solid was suspended in 5 mL CH₂Cl₂, 131 mg PPh₃ (0.50mmol) was added and the reaction mixture was stirred at room temperaturefor 20 min resulting in a clear colorless solution. The solvent wasremoved under reduced pressure and the resulting solid was thoroughlywashed with hexanes to eliminate any excess PPh₃. Finally the solidmaterial was dissolved in CH₂Cl₂ and carefully layered with hexanes.X-ray quality block-shaped colorless crystals of (1). Yield: 60 mg(88%). Anal. Calcd for C₇₈H₆₄N₄Cl₂P₄Ag₂ (1467.93 g mol⁻¹): C, 62.82; H,4.39; N, 3.82. Found: C, 62.73; H, 4.60; N, 3.51. IR (υ_(max), cm⁻¹):3046 (w), 1477 (m), 1432 (s), 1268 (w), 1174 (w), 1093 (m), 1024 (m),952 (m), 815 (w), 742 (s), 692 (s), 615 (w). ¹H NMR (400 MHz, CDCl₃) d(ppm): 7.55 (s, 4H, —CH/pz), 7.69-7.32 (m, 60H, —CH/PPh3). ³¹P NMR(12.35 MHz, CDCl₃, ppm): −30.03 (PPh₃ at −5.89 ppm as reference).

Example 3—Synthesis of [Ag₂(4-Cl-pz)₂(PPh₃)₃] Complex (2)

The [Ag₂(4-Cl-pz)₂(PPh₃)₃] (2) complex was synthesized in the same wayas complex 1, except 1.5 equivalents of PPh₃ was used in this reaction.The solid obtained from the colorless reaction solution was washedthoroughly with hexanes and dried under vacuum. Next, this solid wasdissolved in CH₂Cl₂ and layered with hexanes. Single crystals (ascolorless blocks) of complex 2 were obtained after one week. Yield: 52mg (92%). Anal. Calcd for C₆₀H₄₉N₄Cl₂P₃Ag₂ (1205.64 g C, 59.77; H, 4.10;N, 4.65. Found: C, 59.29; H, 3.99; N, 4.56. IR (υ_(max), cm⁻¹) 3048 (w),1477 (m), 1432 (s), 1268 (w), 1174 (w), 1093 (m), 1024 (m), 952 (m), 817(w), 744 (s), 692 (s), 615 (w). ¹H NMR (400 MHz, CDCl₃) d (ppm): 7.41(s, 4H, —CH/pz), 7.39-7.27 (m, 45H, —CH/PPh₃). ³¹P NMR (CDCl₃, ppm):−27.82 (PPh₃ as reference).

Example 4—Synthesis of [Ag₂(4-NO₂-pz)₂(PPh₃)₄] Complex (3)

The [Ag₂(4-NO₂-pz)₂(PPh₃)₄] (3) complex was prepared by the sameprocedure as complex (1), using [Ag(4-NO₂-pz)]_(n) polymeric precursor(this polymer was obtained from the reaction of Ag(PhCOO) and 4-NO₂-pzHfollowing the same reaction conditions employed to isolate[Ag(4-Cl-pz)]_(n) polymer) and excess PPh₃. Yield: 58 mg (88%). Anal.Calcd for C₇₈H₆₄N₆O₄P₄Ag₂ (1489.04 g mol⁻¹): C, 62.92; H, 4.33; N, 5.64.Found: C, 61.90; H, 4.28; N, 5.66. IR (υ_(max), cm⁻¹): 3050 (w), 1496(m), 1475 (s), 1430 (m), 1392 (s), 1259 (s), 1147 (m), 1089 (m), 1002(m), 852 (w), 811 (w), 742 (s), 690 (s), 601 (s);). ¹H NMR (400 MHz,CDCl₃) d (ppm): 7.91 (s, 4H, —CH/pz), 7.70-7.24 (m, 60H, —CH/PPh3). ³¹PNMR (CDCl3, ppm): −29.40 (PPh₃ as reference.

Example 5—Synthesis of [Ag₂(4-NO₂-pz)₂(PPh₃)₃].4H₂O Complex (4).4H₂O

The [Ag₂(4-NO₂-pz)₂(PPh₃)₃].4H₂O (4).4H₂O complex was prepared by thesame procedure as complex (2), using [Ag(4-NO₂-pz)]n polymeric precursorand 1.5 equivalent of PPh₃. Yield: 52 mg (94%). Anal. Calcd forC₆₀H₄₉N₆O₄P₃Ag₂ (1298.70 g mol⁻1): C, 55.44; H, 4.39; N, 6.46. Found: C,56.22; H, 4.29; N, 6.28. IR (υ_(max), cm⁻¹): 3050 (w), 1479 (s), 1432(m), 1396 (s), 1263 (s), 1151 (m), 1093 (m), 1002 (m), 854 (w), 811 (m),742 (s), 692 (s), 603 (s). ¹H NMR (400 MHz, CDCl₃) d (ppm): 7.81 (s, 4H,—CH/pz), 7.38-7.22 (m, 45H, —CH/PPh3). ³¹P NMR (CDCl₃, ppm): −27.31(PPh₃ as reference).

Example 6—Synthesis of [Ag₂(4-Cl-pz)₂(PTA)₄] PTA Complex (5)

PTA is an attractive auxiliary ligand for various catalytic reactions.PTA is highly biocompatible, has minimal intrinsic toxicity [29], andhas been utilized in the development of transition metal-basedanticancer agents with excellent antiangiogenic and anti-metastaticproperties. The [Ag₂(4-Cl-pz)₂(PTA)₄] PTA (5) complex was prepared bythe same procedure as complex (1), using [Ag(4-Cl-pz)]n polymericprecursor and 4 equivalents of PTA. 10.3 mg (0.101 mmol) 4-Cl-pzH and22.9 mg (0.1 mmol) Ag(PhCOO) were added to 5 mL THF and the reactionmixture was stirred at room temperature for 12 h. After this time, themixture was filtered and the insoluble polymeric [Ag(4-Cl-pz)]n wascollected by filtration and was thoroughly washed with CH2Cl2 to removeany unreacted starting materials. The solid was then dried under vacuum(Yield, 19.5 mg (93%), based on Ag(PhCOO) as a limiting reagent).Subsequently, 19.5 mg (0.093 mmol) of the dry solid was suspended in 5mL CH2Cl2, 58.5 mg PPTA (0.372 mmol) was added and the reaction mixturewas stirred at room temperature for 2 minutes resulting in a clearcolorless solution. Finally, it was allowed to air concentration. X-rayquality block-shaped colorless crystals of 1. Yield: 25 mg (51%). Anal.Calcd. for C₃₆H₆₄N₁₉Cl₂P₅Ag₂ (1787.17 g mol⁻¹): C, 35.90; H, 5.36; N,22.09. Found: C, 36.51; H, 6.04; N, 21.10. IR (υ_(max), cm⁻¹): 3197 (s),2944 (w), 2906 (w), 2275 (w), 1637 (w), 1448 (m), 1411 (s), 1295 (s),1241 (s), 1105 (m), 1037 (s), 1012 (s), 970 (s), 948 (m), 900 (s), 794(m). ¹H NMR (400 MHz, D₂O) d (ppm): 7.67 (s, 4H, —CH/pz), 4.23-3.32 (m,48H, —CH/PPh₃). ³¹P NMR (D₂O, ppm): −178.31 (PTA at −98.51 ppm asreference).

Example 7—Packing Patterns

Analysis of the packing patterns for all five complexes reveals noclassical hydrogen bonding interactions. In case of both (1) and (5), arelatively weak C—H—Cl intermolecular interactions (with C—Cl, 3.580(5)Å, symmetry code: x+1, y, z+1 for (1) and C—Cl, 3.501(4) Å, symmetrycode: x−½, −y+½, z+½ for (5) consolidated their extended structures.

In case of both (3) and (4), a relatively weak C—H—O intermolecularinteractions (with C—O, 3.222(12) Å, symmetry code: x−1, y, z−1 and C—O,3.329(10) Å, symmetry code: −x+1, −y+1, −z+1 for (3) and C—O, 3.461(6)Å, symmetry code: −x+1, −y, −z+1 for (4) consolidated their extendedstructures.

Example 8—X-Ray Data Collection and Structure Refinement

Colorless block-shaped crystals of complexes (1) to (5) were obtained byrecrystallization through diffusion of hexanes into theirdichloromethane (CH₂Cl₂) solutions. In all cases a suitable crystal wasselected and mounted on a Bruker D8 Quest diffractometer equipped withPHOTON 100 detector operating at T=298 K. Data were collected with ωshutter less scan technique using graphite monochromated Mo-Kα radiation(λ=0.71073 Å). The total number of runs and images for data collectionwas based on strategy calculation from the program APEX3 (Bruker) [36].Resolution of θ>26° was achieved in all cases. Cell parameters wereretrieved using the SAINT (Bruker) software [37] and refined using SAINT(Bruker) on 9960 reflections for complexes (1) and (2), 9056 reflectionsfor complex (3), 9879 reflections for complex (4) and 9926 reflectionsfor complex (5). Data reduction was performed using the SAINT (Bruker)software, which corrects for Lorentz and polarization effects. The finalcompleteness was 95.3% for complex (1), 99.7% for complex (2), 99.6% forcomplex (3), 99.4% for complex (4) and 99.2% for complex (5). Multi-scanabsorption corrections were performed on all data sets using SADABS2016/2. The minimum and maximum transmissions for complex (1) were 0.685and 0.746, for complex (2) were 0.695 and 0.745, for complex (3) were0.646 and 0.745, for complex (4) were 0.685 and 0.745 and for complex(5) were 0.646 and 0.745 respectively. The structures for complexes (1)to (4) were solved in the space group P-1 (No. 2) and for complex (5) inC2/c (No. 15) by intrinsic phasing using the ShelXT structure solutionprogram and refined by full matrix least square procedure on F² usingversion 2016/6 of ShelXL. The non-hydrogen atoms were refinedanisotropically in all cases. Hydrogen atom positions were calculatedgeometrically and refined using the riding model. For structures ofcomplexes (1), (3) and (5) only half of the molecule is present in theasymmetric unit, with the other half consisting of symmetry equivalentatoms.

To alleviate the complications related to solvent accessible voidswithin the extended lattice of complex (2), the SQUEEZE operation(included in the PLATON program) was performed with the raw data set andthe structure was refined from the data obtained upon SQUEEZE operation.Despite of several attempts, in case of complex (4), an accurateposition of the hydrogen atoms for the lattice water molecules was notachieved. Therefore no hydrogen was added on those oxygen atoms.

All calculations and molecular graphics were preformed using eitherSHELXTL 2014 or Olex2 programs. Crystal data and structure refinementparameters are listed in Table 1. CCDC 1825643 (complex 1), CCDC 1825644(complex 2), CCDC 1825646 (complex 3), CCDC 1825647 (complex 4) and CCDC1825645 (complex 5) contain the supplementary crystallographic data.These data can be obtained from The Cambridge Crystallographic DataCenter via www.ccdc.cam.ac.uk/data_request.cif.

TABLE 1 Crystal data and structure refinement parameters for complexes(1), (2), (3), (4) and (5) (1) (2) (3) (4)).4H₂O (5) FormulaC₇₈H₆₄Ag₂N₄Cl₂P₄ C₆₀H₄₉Ag₂N₄Cl₂P₃ C₇₈H₆₄Ag₂N₆O₄P₄ C₆₀H₅₇Ag₂N₆O₈P₃C₃₀H₅₂Ag₂N₁₆Cl₂P₄ D_(calc.)/g cm⁻³ 1.434 1.335 1.443 1.374 1.722 μ/mm⁻¹0.80 0.86 0.72 0.76 1.31 Formula 1467.85 1205.58 1488.97 1298.70 1047.39Weight Color Colorless Colorless Colorless Colorless Colorless ShapeBlock Block Block Block Block T/K    298 (2)    298 (2)    298 (2)   298(2)    298 (2) Crystal Triclinic Triclinic Triclinic TriclinicMonoclinic System Space Group P-1 P-1 P-1 P-1 C2/c a/Å 12.3970 (7)11.9900 (6) 12.4684 (8) 12.0477 (6)   22.3522 (18) b/Å 12.6513 (7)13.4064 (7) 12.7646 (9) 13.9333 (6)   7.6984 (6) c/Å 13.4150 (7) 20.5531 (11)  13.4383 (10) 20.1754 (10)  25.899 (2) α/°  97.372 (2) 93.164 (2)  98.117 (2) 81.223 (1) 90 β/° 116.099 (1)  93.563 (2)116.230 (2) 84.884 (1) 114.962 (1) γ/° 108.552 (2) 114.083 (1) 108.435(2) 68.853 (1) 90 V/Å³  1700.01 (16)  2998.6 (3)  1714.0 (2) 3119.5 (3) 4040.2 (6) Z 1 2 1 2 4 Wavelength/Å 0.71073 0.71073 0.71073 0.710730.71073 Radiation Mo-Ka Mo-Ka Mo-Ka Mo-Ka Mo-Ka type 2θ_(min)/° 6.006.00 5.80 5.80 6.20 2θ_(max)/° 56.60 52.80 53.00 52.60 52.80 Measured28423 55827 31251 57586 38314 Refl. Independent 8045 12266 7051 126234113 Refl. Reflections 6244 8456 5461 10176 3876 Used R_(int) 0.0320.043 0.051 0.024 0.019 Parameters 406 622 424 712 244 ^(a)GooF 1.0501.040 1.070 1.040 1.190 ^(c)wR₂ 0.083 0.107 0.154 0.129 0.073 ^(b)R₁0.037 0.045 0.058 0.039 0.029 ^(a)GOF = [Σ[w(F_(o) ² − F_(c) ²)²]/(N_(o)− N_(v))]^(1/2) (N_(o) = number of observations, N_(v) = number ofvariables). ^(b)R₁ = Σ||F_(o)| − |F_(c)||/Σ|F_(o)|. ^(c)wR₂ = [(Σw(F_(o)² − F_(c) ²)²/Σ|F_(o)|²)]^(1/2)

Example 9—X-Ray Crystallographic Analyses

Single crystal X-ray crystallographic analyses revealed the molecularstructures for all complexes (1) to (5). Among them the perspective viewfor the structures (1), (2), (3), (4) and (5) are depicted in FIGS. 4,5, 6, 7, and 8 and selected metric parameters are listed in Tables 2, 3,4, and 5. In case of complexes (1), (3) and (5) only one half of theformula is present in the asymmetric unit, with other half consisting ofsymmetry equivalent atoms.

The X-ray structures of complexes (1), (3) and (5) (FIGS. 4, 6 and 8,respectively) revealed that in all three cases the two Ag(I) centerswere equivalent and each metal center resided in a distorted tetrahedralcoordination environment. To assess the distortion from the idealtetrahedral geometry around the Ag centers, a simple index (τ4)developed by Houser and co-workers was calculated for complexes (1) and(5). This simple index is unity for a perfect tetrahedron and zero forsquare planar geometry. The τ4 values for complexes (1) and (5) were0.89 and 0.78 respectively suggesting the coordination environmentaround the Ag centers was distorted tetrahedral in these complexes. Twoμ-pyrazolates bridged the Ag(I) centers and the other two coordinationsites of each Ag(I) center were occupied by two PPh₃ ligands. The two Agatoms along with four N atoms (Ag1, Ag1a, N1, N2, N1a, N2a) of thebridging pyrazolates formed a centrosymmetric six-membered ring in achair conformation, with one Ag-atom on either side of the planesdefined by the pyrazolate rings and at a Ag . . . Ag distance of4.209(4) Å (1) and 4.205(3) Å (5). This twisted metallacyclicconformation is rare in literature, but not unprecedented. Althoughcomplexes (1) and (5) are structurally similar, careful scrutiny of themetric parameters revealed certain differences (Table 1): The averageAg—N bond length in complex (1), 2.319(2) Å, was shorter than that incomplex (5) (2.367(3) Å); The P—Ag—P angle in complex (1), 118.82(2)°,was noticeably more acute than the corresponding 140.43(3)° angle incomplex (5) and the same applied to the N—Ag—N bond angles, 98.00(7)° incomplex (1) and 103.37(9)° in complex (5), the differences attributed tothe steric bulk of PTA compared to PPh₃. Consequently, the averageN1-Ag1-P1 angle in complex 1 (109.32(6)° was more obtuse than in complex5 (101.80(7)°). The average Ag1-P1 bond lengths for these two complexes,2.4921(7) Å complex (1) and 2.4972(8) Å complex (5), respectively, wereindistinguishable.

TABLE 2 Selected bond distances (Å) and angles (°) for complexes (1) and(5). Complex (1) Complex (5) Ag1—P1 2.5003(7) 2.5345(8) Ag1—P2 2.4838(7)2.4598(7) Ag1—N1 2.285(2) 2.336(2) Ag1—N2a 2.353(2) 2.398(3) P2—Ag1—P1118.82(2) 140.43(3) N1—Ag1—P2 110.20(6) 109.52(7) N1—Ag1—P1 115.09(6)100.00(7) N1—Ag1—N2a 98.00(7) 103.37(9) N2a—Ag1—P2 105.32(6) 105.46(7)N2—Ag1—P1 106.68(6) 92.23(7) The a-labeled atoms were generated by the−x + 1, −y, −z + 1 symmetry operation

The crystal structure of complex (2) revealed an unsymmetrical dinuclearcomplex (FIG. 4). In this case, the coordination geometry of one Ag(I)center was distorted tetrahedral, whereas that of the other Ag(I) centerwas approximately triangular planar.

TABLE 3 Selected bond distances (Å) and angles (°) for complex (2)Complex (2) Ag1—P1 2.4852(9) Ag2—P3 2.3735(11) Ag1—P2 2.4751(9) Ag2—N42.204(3) Ag1—N1 2.260(3) Ag2—N2 2.246(3) Ag1—N3 2.361(3) P2—Ag1—P1121.03(3) N4—Ag2—P3 130.82(9) N1—Ag1—P2 121.43(8) N4—Ag2—N2 108.63(12)N1—Ag1—P1 107.29(8) N2—Ag2—P3 120.06(8) N1—Ag1—N3 95.28(11) N3—Ag1—P2102.40(8) N3—Ag1—P1 104.14(8)

In complex (2), the average Ag—N bond length of 2.311(3) Å at thetetrahedral site was comparable to the corresponding distance in complex(1), while that of the trigonal planar site, 2.225(3) Å, wasconsiderably shorter. The P1-Ag1-P2 angle (tetrahedral site) of121.03(3)° and the N1-Ag1-N3 angle of complex (2) (95.28(11)° werecomparable to the corresponding values in complex (1). However, theN4-Ag2-N2 angle was noticeably larger)(108.63(12)°, consistent with theplanar coordination sphere around Ag2 center. A structurally similarcomplex as complex (2) derived from unsubstituted pyrazole, namely,[Ag₂(μ-pz)₂(PPh₃)₃] had been reported earlier. In this case the Ag—Nbond length at the tetrahedral and the triangular planar sites were2.309(2) and 2.206(2) Å, respectively, comparable to the values observedin complex (2). The bond angles for both tetrahedral and the planarsites in [Ag₂(μ-pz)₂(PPh₃)₃] and complex (2) were rather similar. The Ag. . . Ag distance in complex (2) was 3.707(3) Å almost identical withthat in [Ag₂(μ-pz)₂(PPh₃)₃] (3.706(1) Å).

TABLE 4 Selected bond distances (Å) and angles (°) for (3). Complex (3)Ag1—P1 2.5017(12) Ag1—P2 2.4868(12) Ag1—N1 2.307(4) Ag1—N2a 2.378(4)P2—Ag1—P1 120.75(4) N1—Ag1—P2 110.74(11) N1—Ag1—P1 114.88(11) N1—Ag1—N2a96.10(14) N2a—Ag1—P2 105.86(11) N2—Ag1—P1 104.75(10)

Complex (3) (FIG. 6) was structurally quite similar to complex (1) (FIG.4). The two Ag atoms in this structure also formed a twistedmetallacycle with a Ag . . . Ag distance of 4.305(4) Å. The average Ag—Nbond length in complex (3), 2.343(4) Å was similar to that in complex(1) (2.319(2) Å). The P—Ag—P angle in complex (3), 120.75(4) wasconsistent with that found in complex (1)(118.82(2)°.

TABLE 5 Selected bond distances (Å) and angles (°) for (4) Complex (4)Ag1—P1 2.4694(8) Ag2—P3 2.3789(8) Ag1—P2 2.4948(8) Ag2—N5 2.187(3)Ag1—N1 2.373(3) Ag2—N2 2.278(3) Ag1—N4 2.355(3) P1—Ag1—P2 124.62(3)N5—Ag2—P3 144.74(8) N4—Ag1—P2 105.26(8) N5—Ag2—N2 105.06(11) N4—Ag1—P1115.20(7) N2—Ag2—P3 109.14(8) N4—Ag1—N1 97.24(11) N1—Ag1—P2 108.56(8)N1—Ag1—P1 102.28(8) The a-labeled atoms were generated by the −x + 1,−y + 2, −z + 1 symmetry operation

The two Ag atoms in complex (4) (FIG. 7) exhibited the same coordinationgeometry as in complex (2) (FIG. 5) with an average Ag—N bond length of2.364(3) Å at the tetrahedral site, slightly longer than thecorresponding distance in 2, and 2.233(3) Å at the trigonal planar site.At the tetrahedral site of complex (4), the P1-Ag1-P2 and N1-Ag1-N4angles were 24.62(3)° and (97.24(11)°, respectively, comparable to thecorresponding values in complex (2).

Example 10—NMR Spectra

In most cases the solution structure of coordination complexes withsubstitutionally labile ligands (like PPh₃ and PTA in the present case)was different from the solid-state structure. Therefore, ³¹P {¹H} NMRstudies were performed with complexes (1) to (5) to gain an insight inpossible solution characteristics of these complexes. Due to differencein solubility, ³¹P NMR spectroscopy for complexes (1) to (4) wasperformed in CDCl₃ solutions, while the same for complex (5) was carriedout in D₂O (due to poor solubility in CDCl₃ or CD₂Cl₂). For the formercomplexes PPh₃ was used as standard and PTA was employed for the latterone. The substantial kinetic lability of the phosphine ligands oftenleads to rapid ligand exchange in solution at ambient temperature at theNMR time scale. Indeed, ³¹P NMR spectra of complexes (2) and (4),recorded in the 275-295 K temperature range, consisted of a singleresonance resulting from the convergence of the two magneticallynon-equivalent PPh₃ ligands, revealing the fluxional behavior of thesecomplexes in solution. The comparative ³¹P NMR spectra are shown in FIG.3. The room temperature ¹H-NMR spectra of complexes (2) and (4) alsoshowed a single resonance for the pyrazole H³ and H⁵ protons, inagreement with the ³¹P NMR results.

Example 11—Antibacterial Activity

A skin and soft tissue infection (SSTI) model was used to study theantibacterial properties of three silver complexes. In this model, twolayers of agar were employed to mimic a tissue infection with a nutrientrich bottom and the soft-top layer to allow for even dispersal ofbacteria. The top soft agar acts like the skin and as bacteria grow theymigrate towards the bottom nutrient layer mimicking an infected tissue.Frozen samples of Pseudomonas Aeruginosa were thawed and streaked on anagar plate. A single colony was selected and grown in Luria Broth (LB)for 18 h. The suspension was diluted with LB until an A₆₀₀ (absorbance)of 0.5 was reached. The soft agar bacterial suspension was prepared byadding 120 mL of the above solution to 100 mL 0.8% (w/v) agar with 1%NaCl, which had been autoclaved and cooled to 45° C. before addition.The suspension was gently vortexed and 8 mL was spread evenly on thesurface of 20 mL of a 1.5% (w/v) TSB agar plate (100−15 mm²) and allowedto solidify. The plates were incubated for 2 h at 37° C. in order forthe bacteria to reach log phase before bactericidal experiments wereperformed.

Blank KBr pellets (typically utilized for recording IR spectra) and KBrpellets with 2% (w/w) of three complexes (1), (2) and (5), each weighingbetween 34 and 46 mg, were evenly mulled and pressed with a two-tonload. Three blanks, containing PTA, or 4-Cl-pzH, or PPh₃, were alsoprepared in the same manner. Pellets were carefully placed on the plates(see Example 8) after 2 h of incubation at 37° C. Visible circular zonesof bacterial clearing (zone of inhibition) around complex (5) and AgNO₃pellets were observed after incubation at 37° C. for 18 h (FIG. 24).Complex (5) and AgNO₃ pellets revealed comparable bacterial clearanceeven though the KBr pellet with complex (5) contained less silver perweight than the AgNO₃ pellet. This superior growth inhibitoryperformance of complex (5) could in part be due to marginalantibacterial effects of ligands (PTA and 4-Cl-pzH), which showed muchsmaller but still visible zones of bacterial growth inhibition. Theplates containing complexes (1) and (2) exhibited minimal bacterialclearance while PPh₃ and KBr (bottom row) showed no zone of inhibition.Also the % (w/w) of Ag and concentration of bio-active silver in platesa), b), e) and f) are listed in Table 6.

The antibacterial efficacy was highly dependent on efficient cellularuptake of the compound. A slightly electronegative surface potential ofbacterial cell walls allowed cationic complexes to penetrate relativelyeasily compared to their neutral analogues All silver(I) complexes wereneutral and thereby lacking this advantage, as could be seen from themarginal antibacterial effects of complexes (1) and (2) (FIG. 24).However exceptional growth inhibition of P. aeruginosa by complex (5)could be attributed to enhanced cellular uptake of this complexfacilitated by the PTA moieties due to their optimal lipophilicity. Thebridgehead N atoms of the PTA moiety become protonated under certainphysiological conditions and can thereby contribute towards facilecellular uptake and thereafter retention of its complexes. In fact, theadamantane motif can be used in as a “lipophilic bullet” to allow rapidinternalization into a variety of cells and tissues. It is interestingto note that the majority of Ag-PTA compounds are polymeric and theirbridgehead N atoms also engage in bonding with silver. Therefore, in thesubject invention, new discrete dinuclear silver (I) pyrazolidocomplexes are provided, where PTA is directly coordinated to Ag(I)centers and no further polymeric growth through the N atoms of the PTAmoiety occurs.

TABLE 6 Concentration and % (w/w) Ag in each pellet (before diffusion).Sample Conc of Ag in the pellet (M) % (w/w) Ag in the pellet 1 1.03548 ×10⁻⁶ 0.29394 2 1.26075 × 10⁻⁶ 0.35789 5 1.45127 × 10⁻⁶ 0.41197 AgNO₃4.47401 × 10⁻⁶ 1.27003

A previously developed soft tissue infection (SSTI) model has beenutilized for the present antibacterial studies. This model mimics theskin in which steady penetration of bacteria to the deeper layer hasbeen achieved using a two-layer agar system that has a soft, evenlydispersed bacterial lawn on the top and a nutrient-rich bottom layer(see Example 11). The gradient causes the bacteria to slowly travel fromthe slender top layer to the nutrient-rich bottom layer, much alike whatwould occur in a typical skin infection.

Three complexes (1), (2) and (5) along with 4-Cl-pzH and two thephosphine ligands (PPh₃ and PTA) have been assessed for theirbactericidal efficacy against a Gram-negative aerobicgamma-proteobacterium namely, Pseudomonas aeruginosa. This bacterium isone of the most notorious contributors to the nosocomial infectionsaround the World. During pre-penicillin G era, Staphylococcus aureusused to be the most common pathogen responsible in burn woundinfections. Although this Gram-positive bacterium still remains one ofthe sources for such infections, in recent times P. aeruginosa has beenrecognized as major cause of burn wound infections in hospitals. Apartfrom P. aeruginosa, fatal burn wound sepsis can also be instigatedthrough infections caused by S. aureus and A. Baumannii. However, a veryrecent study revealed that P. aeruginosa infections have the most severeconsequences towards burn injuries and often lead to mortality. In thesame study, the effect of the burn wound exudates from a group ofpatients has been investigated on virulence of several pathogens(including S. aureus and A. Baumannii). Interestingly among all thepathogens, P. aeruginosa exclusively exhibited normal proliferationwithin these exudates. In contrast, kinetic studies clearly delineatedgrowth inhibitory effect of the burn wound exudates for other pathogens.Development of therapeutics to combat this nosocomial pathogen deservesurgent attention as with time these microorganisms have developedincreasing resistance to antibiotics, particularly in immunocompromisedand cystic fibrosis patients.

Silver nitrate and silver sulfadiazine (SSD) are two widely used topicalantimicrobial agents for burn wound infections. However, silver nitrateand SSD topical therapeutics suffer from serious limitations: rapidreaction of silver nitrate with biological chloride ions forms insolublesilver chloride and thereby requires continuous administration with theocclusive dressings. Also, although dissociation of silver ions from ofSSD is slower compared to that in silver nitrate, poor dermalpenetration of this topical agent limits its efficacy on severe burnwound sites. Moreover, a nephrotic syndrome is prevalent within patientsreceiving topical SSD therapy, due to an allergic reaction associatedwith the sulfadiazine component.

Therefore, the subject invention provides silver pyrazolido complexes asalternative topical therapeutics against P. aeruginosa infections asdemonstrated utilizing a SSTI model. The lability of Ag—P bonds, as hasbeen discussed in detail earlier by others, is evident by the NMRspectroscopic behavior of, e.g., complexes (2) and (4). Such labilityinduces the release of bioactive Ag to the wound sites and, withoutwanting to be bound by theory, it is believed that the induced releaseof Ag to the wound is a contributing factor towards the unexpectedsuperior antibacterial activity of complexes of the subject invention,e.g., complex (5).

Therefore, five examples of dinuclear silver(I) pyrazolido complexes (1)to (5) are provided, which complexes are derived from two 4-substitutedpyrazole ligands that have been synthesized and structurallycharacterized according to methods of the subject invention. The highlywater-soluble complex (5) incorporating a PTA co-ligand was shown tohave unexpected excellent antibacterial activity in a SSTI model againstP. aeruginosa. The zone of inhibition, which inhibition could bebactericidal or bacteriostatic, in the bacterial culture caused byapplication of complex (5) is comparable to that for AgNO₃. The 4-Cl-pzHligand shows marginal growth inhibition, in line with the knownantimicrobial properties of pyrazoles. The relatively lower efficacy ofthe two organosoluble complexes (1) and (2) is tentatively attributed toinferior cellular uptake considering all these complexes are neutral incharge.

In contrast, the highly lipophilic nature of the PTA motif, in case,e.g., of complex (5) overwhelms the disadvantage of its dinuclear Ag(I)pyrazolido complex, including the complex being neutral, which isconsidered unfavorable towards cellular internalization compared to apositively charged analogue.

Furthermore, complex (29) having only a single PTA bound by each silveratom showed surprising superior effectiveness, even compared to thehighly anti-bacterial complex (5). Both complex (5) and complex (29)were comparatively tested against P. aeruginosa. The ligands of thesecomplexes displayed no antimicrobial activity and both complexes werereadily soluble in water and several bacterial growth media. Complex (5)exhibited a dose-dependent eradication of the bacterial culture with anIC₅₀>2000 μg/mL.

In contrast, complex (29) was much more potent and upon application ofsimilar dosages as in case of complex (5) demonstrated already almost90% eradication of the bacterial culture at the lowest concentration of31.25 μg/mL tested.

Therefore, while AgNO₃ and AgSD are regularly utilized in burn victimwards of hospitals for wound healing purposes, the subject inventionprovides novel biocompatible silver complexes that have significantlysuperior effectiveness against bacteria including P. aeruginosa comparedto commonly used compounds including, but not limited to, AgNO₃ andenable a slow and sustainable delivery of the bio-active silver to thesite of interest. Moreover, the biocompatible ligands and co-ligands ofthe complexes of the subject invention have low toxicity upon therelease of Ag⁺ under physiological environmental conditions.

Advantageously, silver-based dinuclear Ag(I) pyrazolido complexes of thesubject invention are a new generation of chemotherapeutics to combatantibiotic resistance and treat bacterial, fungal and viral infections.While the toxicity profile of many nanomaterials towards human healthand environment prevents their ready use in vivo, the dinuclear Ag(I)pyrazolido complexes of the subject invention, due to their optimizedcombination of lipophilic and hydrophilic properties, show unexpectedexcellent cellular uptake, reduced toxicity and unexpected excellentantibacterial, antifungal and antiviral potency.

We claim:
 1. A dinuclear silver (I) pyrazolido complex having thefollowing structure (C)

wherein R¹ and R² are each independently selected from the groupconsisting of 1,3,5-triaza-7-phosphaadamantane (PTA), phosphaadamantane(PA), (4-sulfophenyl)diphenylphosphine (SDPP), phenanthrenediphenylphosphine (PDPP),8-((4-phosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PMBODIPY),8-((4-phosphino)phenyl)-4,4-diethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PEBODIPY),and8-((4-phosphino)phenyl)-4,4-diphenyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene(PPBODIPY); m is an integer selected from the group consisting of 0 and1; n is an integer selected from the group consisting of 0 and 1; R³,R⁴, and R⁵ are each independently selected from the group consisting ofhydrogen, halogen, hydroxyl, nitro, alkyl, alkenyl, aryl, formyl,acetyl, hydroxyalkyl, halogen substituted alkyl, halogen substitutedaryl, halogen substituted sulfuryl, imine-linked PTA, amine-linked PTA,imine-linked adamantane, amine-linked adamantine, imine-linkedtriphenylphosphine, and amine-linked triphenylphosphine; or apharmaceutically acceptable salt thereof; wherein the dinuclear silver(I) pyrazolido complex is selected from the group consisting of:


2. A composition comprising a dinuclear silver (I) pyrazolido complexaccording to claim
 1. 3. The composition according to claim 2, furthercomprising one or more ingredients selected from anti-bacterial agents,anti-viral agents, fungicidal agents, anesthetic agents, buffers, andpharmacological excipients.